It has long been known that articular chondrocytes maintain a stable phenotype through life, whereas growth plate chondrocytes modulate their phenotype, undergo maturation and hypertrophy, and are replaced by bone. However, it is still largely unclear how chondrocytes are able to maintain or modulate their phenotype to exert those fundamentally distinct functions. Runx2 and PPARgamma are two transcription factors that have received a great deal of attention recently. Runx2 is up-regulated in, and required for, chondrocyte hypertrophy and ossification, and PPARgamma is expressed in human articular cartilage and exerts anti-inflammatory roles. We have now found that both factors are expressed in epiphyseal and immature growth plate chondrocytes. Runx2 over-expression accelerates maturation, whereas over-expression of a dominant-negative (DN) Runx2 blocks maturation and leads to chondrocyte de-differentiation. The latter is seen also in chondrocytes isolated from Runx2-/- mouse embryos. In contrast, PPARgamma over-expression blocks maturation, while DN-PPARgamma over-expression favors maturation. Additional in vitro and in vivo data indicate that expression of Runx2 and PPARgamma is tightly coordinated and that the two genes have interdependent roles in chondrocytes. Our central hypotheses are: (a) Continuous and relatively low expression of Runx2 and PPARgamma is needed for a stable chondrocyte phenotype; and (b) Runx2 up-regulation and PPARgamma down-regulation lead to chondrocyte maturation and hypertrophy. We will test these hypotheses by analyzing pre-natal and post-natal patterns of expression and activity of Runx2 and PPARgamma in articular and growth plate cartilages and cells. We will determine consequences of experimental manipulation of Runx2 and PPARgamma expression and activity in vitro and in vivo, including conditional ablation of each gene in chondrocytes at prenatal and/or postnatal points. Molecular mechanisms of regulation of Runx2 and PPARgamma will be tested, including possible direct protein-protein interactions resulting in complexes with altered trans-activating properties. The results of the project will shed novel and important insights into the molecular regulation of chondrocyte phenotype, and will suggest new ways to improve cell and tissue function during natural aging and pathological conditions of the skeleton and joints.